Labelled Adrenomedullin Derivatives and Their Use for Imaging and Therapy

ABSTRACT

The present invention relates to an adrenomedullin derivative including an adrenomedullin peptide chelated with at least one active agent. Examples of active agents include a paramagnetic element, a radioactive element and a fibrinolytic agent, among others. Paramagnetic agents have a distribution that is relatively easily shown through Magnetic Resonance Imaging (MRI). Radioactive agents have applications in imaging and delivery of radiations, depending on the specific element included in the active agent. Delivery of fibrinolytic agents mainly to a specific organ, such us for example to the lungs, allows to substantially improve the specificity and efficacy of thrombolytic therapy by allowing local delivery of the fibrinolytic agent, thereby reducing the risks of major bleeding in the therapy of the organ. If the organ is the lungs, a non-limiting example of pathology treatable with the fibrinolytic is pulmonary embolus.

This application claims priority from U.S. Provisional PatentApplication Ser. No. 60/573,334 filed May 24, 2004.

FIELD OF THE INVENTION

The present invention relates to the use of labelled compounds forimaging or therapy. More specifically, the present invention isconcerned with labelled adrenomedullin derivatives and their use forimaging and therapy.

BACKGROUND OF THE INVENTION

A currently existing agent for the clinical imaging of pulmonarycirculation is ^(99m)Tc-labelled albumin macroaggregates. This agent isused for the diagnosis of physical defects of the circulation due topulmonary embolus. This agent is larger than small pulmonary vessels.Accordingly, further to being injected in an animal, this agent istrapped by these small pulmonary vessels, which enables externaldetection.

Important limitations of albumin macroaggregates include the inabilityto image the small pulmonary circulation beyond the point ofobstruction. This limits the sensitivity of this substance to detectsmall vascular defects. Also; there are potential infectious risks sincealbumin macroaggregates are derived from human albumin. Additionally,albumin macroaggregates are unable to detect functional (biological)defects of the pulmonary circulation since their retention is uniquelydependent on physical characteristics of the vessels.

There also exist compounds that have an affinity for particular organs,such as for example adrenomedullin (AM). AM is a 52-amino-acidmultifunctional regulatory peptide highly expressed in endothelial cellsand widely distributed in various tissues [1,2]. The structure of AM iswell conserved across species, with only six substitutions and twodeletions in the rat [rAM(1-50)] compared with the human [hAM(1-52)][3]. AM possesses structural homology with CGRP (calcitonin gene-relatedpeptide), making it a member of the calcitonin/CGRP/amylin family(CT/CGRP/AMY peptide family.

The biological activities of AM are mediated by receptors composed oftwo essential structural components: a seven-transmembrane protein, thecalcitonin receptor-like receptor (CRLR), and a single transmembranedomain termed RAMP (receptor-activity-modifying protein) [4,5]. Theassociation of CRLR/RAMP1 represents the CGRP1 receptor and is notspecific to AM. At the opposite, a specific AM receptor comes from thecoupling of CRLR/RAMP2 or CLRL/RAMP3 [6]. This specific AM receptor canbe blocked by the C-terminal AM fragment [hAM(22-52)] [7].

A biological action of AM is a potent hypotensive effect. The systemichypotensive action of AM can however be reduced and sometimes abolishedafter intravenous compared with intra-arterial infusion [8], suggestingthat the lungs have a potential to clear circulating AM and modulate itscirculating levels. Many studies have confirmed that AM is cleared bythe pulmonary circulation [9-12]. However, the relative contribution ofthe lungs to AM clearance in comparison with other organs has not beensystematically evaluated and, more specifically, single-pass pulmonaryclearance of AM has not been quantified in vivo.

Against this background, there exists a need in the industry to providenovel compounds having an affinity for the lungs, and more specificallyto provide such compounds suitable for use in therapy and imaging.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

OBJECTS OF THE INVENTION

An object of the present invention is therefore to provide novelcompounds having an affinity for the lungs.

SUMMARY OF THE INVENTION

In a first broad aspect, the invention provides an adrenomedullinderivative comprising an adrenomedullin peptide chelated to at least oneactive agent. For example, the adrenomedullin derivative comprisesadrenomedullin having the sequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID 1), or a fragment thereof. Examples of active agents includeactive agents comprising at least one paramagnetic element, activeagents comprising at least one radioactive element, and fibrinolyticagents, among others.

The biodistribution, pharmacokinetics and multi-organ clearance of AMwere evaluated in rats and its single-pass pulmonary clearance wasmeasured in dogs by the indicator-dilution technique. Intravenouslyadministered ¹²⁵I-rAM(1-50) [rat AM(1-50)] was rapidly cleared followinga two-compartment model with a relatively rapid distribution half-lifeof 2.0 min [95% Cl (confidence interval), 1.98-2.01] and an eliminationhalf-life of 15.9 min (95% Cl, 15.0-16.9). The lungs retained most ofthe injected activity with evidence of single-pass clearance, sinceretention was lower after intra-arterial (13.5±0.6%) compared withintravenous (30.4±1.5%; P<0.001) injection. Lung tissue levels of totalendogenous AM were about 20-fold higher than in other organs with nodifference in plasma levels across the pulmonary circulation. In dogs,there was 36.4±2.1% first-pass unidirectional extraction of¹²⁵I-rAM(1-50) by the lungs that was reduced to 21.9±2.4% after theadministration of unlabelled rAM(1-50) (P<0.01). Extraction was notaffected by calcitonin gene-related peptide administration (40.6±2.9%),but slightly reduced by the C-terminal fragment of human AM(22-52)(31.4±3.3%; P<0.01). These data demonstrate that the lungs are a primarysite for AM clearance in vivo with approximately 36% first-passextraction through specific receptors. This suggests that the lungs notonly modulate circulating levels of this peptide, but also represent oneof its primary targets.

In addition, a chelated human AM derivative (hAM-1-52) was developedusing diethylenetriaminepentaacetic acid (DTPA) radiolabelled with^(99m)Tc. The biodistribution, plasma kinetics and utility of^(99m)Tc-DTPA-hAM1-52 as a pulmonary vascular imaging agent wereevaluated.

After HPLC purification, the radiochemical purity of^(99m)Tc-DTPA-hAM1-52 was 92.0±2.3%. A hemodynamically inactive dose ofthe compound was intravenously injected to anesthetized dogs (n=6) andthe tracer activity serially determined in blood samples as well as invarious regions of interest using external detection with a gammacamera. In additional experiments, the capacity to image perfusiondefects was evaluated after selective surgical pulmonary lobectomy.

^(99m)Tc-DTPA-hAM1-52 was relatively rapidly cleared from plasmafollowing a two-compartment model with a relatively rapid distributionhalf-life of 1.75 min (95% confidence interval, Cl: 1.31-2.65). Thelungs retained most of the injected activity after 30 minutes(27.0±2.7%, p<0.001), as compared to kidneys (19.2±3.1%), liver(11.7±1.4%), heart (7.2±2.0%), bladder (5.7±1.7%) and gallbladder(1.0±0.4%). Lung retention was mildly reduced with time but sustained upto 4 hours after the injection (15.8±2.3%), while uptake progressivelyincreased in the bladder (26.8±4.3%) and gallbladder (5.50±2.6%). Afterselective pulmonary lobectomy, anatomically corresponding perfusiondefects were easily detectable by external imaging. Therefore, chelatedAM derivatives display important and extended lung retention and arepromising new agents for pulmonary vascular imaging. Their use can imageanatomical perfusion defects, but also has the inherent potential forthe detection functional vascular perfusion abnormalities.

Advantageously, AM has a potential to image the small pulmonarycirculation. Also by its nature, AM allows a functional imaging of thepulmonary circulation, which is advantageous for some diagnosticpurposes.

In addition, AM is a physiological endogenous peptide used in traceramounts. At those concentrations, AM is devoid of any significantbiological activity. Furthermore, risks of infection are reduced inusing AM instead of albumin macroaggregates for pulmonary imaging.

In a second broad aspect, the invention provides a method of detectingthe presence or absence of pulmonary embolus in a mammal. The methodcomprises:

-   -   administering to the mammal a labelled adrenomedullin derivative        in an amount and for a duration effective to achieve binding        between the labelled adrenomedullin derivative and pulmonary        adrenomedullin-receptor-bearing cells;    -   generating an image of the distribution of the labelled        adrenomedullin derivative in the lungs of said mammal; and    -   detecting the presence or absence of pulmonary embolus.

In a third broad aspect, the invention provides a method of detectingthe presence and density of adrenomedullin receptor-bearing cells in amammal comprising:

-   -   administering to the mammal a labelled adrenomedullin derivative        for a time and under conditions effective to achieve binding        between the labelled adrenomedullin derivative and        adrenomedullin-receptor-bearing cells, and    -   determining the distribution of the labelled adrenomedullin        derivative for a time and under conditions effective to obtain        an image of the mammal.

In a fourth broad aspect, the invention provides a method of deliveringat least one active agent to pulmonary adrenomedullin-receptor-bearingcells in a mammal, the method comprising administering to the mammal anadrenomedullin derivative chelated to the active agent in an amount andfor a duration effective to achieve binding between the adrenomedullinderivative and the pulmonary adrenomedullin-receptor-bearing cells.

If the active agent includes a fibrinolytic agents, chelation offibrinolytic agents to AM derivatives has the potential to substantiallyimprove the specificity and efficacy of thrombolytic therapy by allowinglocal delivery of the fibrinolytic agent, thereby reducing the risks ofmajor bleeding in the therapy of pulmonary embolus.

In a fifth broad aspect, the invention provides a use of labelledadrenomedullin derivatives to image the lungs of a mammal.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of preferred embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 illustrates the plasma kinetics of ¹²⁵I-rAM(1-50) after a singleintravenous injection in a rat;

FIG. 2 illustrates the Biodistribution of ¹²⁵I-rAM(1-50) afterintravenous injection in rats (t P<0.001 compared with the lungs; *P<0.005 and § compared with control, n=10/group);

FIG. 3 illustrates the pulmonary retention of ¹²⁵I-rAM(1-50) in ratsafter intravenous and intra-arterial injection († P<0.005 and § P<0.001compared with venous injection, n=10/group);

FIG. 4 illustrates an example of indicator-dilution curve outflowprofiles in the canine pulmonary circulation in control conditions (A)and after injection of unlabelled rAM(1-50) (B), Insets showing thenatural log ratio curves of the tracers (FR is fractional recovery ofeach tracer);

FIG. 5 illustrates the plasma kinetics of ^(99m)Tc-DTPA-hAM1-52 aftersingle intravenous injections in dogs, the data being fitted with atwo-phase exponential decay equation (the inset shows a logarithmicscale, n=6/group);

FIG. 6 illustrates the biodistribution of ^(99m)Tc-DTPA-hAM1-52 afterintravenous injection in dogs (†P<0.005 vs. 30 minutes; *P<0.001 vs. 30minutes; §P<0.001 vs. lungs, n=6/group);

FIG. 7 illustrates the dynamic biodistribution of ^(99m)Tc-DTPA-hAM1-52after intravenous injection in dogs (n=6/group);

FIG. 8 is a gamma camera image of a dog's thorax obtained further to aninjection of ^(99m)TC marked AM; and

FIG. 9 illustrates selective surgical pulmonary lobectomy effects on^(99m)Tc-DTPA-hAM1-52 perfusion in dog as imaged through a gamma camera:(A) anterior view, (B) oblique view, a wedge shaped perfusion defectbeing indicated by an arrow and delimitated by dotted lines.

DETAILED DESCRIPTION

The present invention relates to the use of an adrenomedullin derivativeincluding an adrenomedullin peptide chelated to at least one activeagent. For example, the adrenomedullin peptide comprises adrenomedullinhaving the sequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:1), or a fragment thereof. This adrenomedullin peptidecorresponds to amino acids 1-52 of human adrenomedullin. Additionally,fragments of adrenomedullin correspond to shorter peptide sequences,such as amino acids 1-50 of rat adrenomedullin.

Examples of active agents include a paramagnetic element, a radioactiveelement and a fibrinolytic agent, among others. Paramagnetic agents havea distribution that is relatively easily shown through MagneticResonance Imaging (MRI). Radioactive agents have applications in imagingand delivery of radiations, depending on the specific element includedin the active agent. Delivery of fibrinolytic agents mainly to aspecific organ, such as for example to the lungs, allows tosubstantially improve the specificity and efficacy of thrombolytictherapy by allowing local delivery of the fibrinolytic agent, therebyreducing the risks of major bleeding in the therapy of the organ. If theorgan is the lungs, a non-limiting example of pathology treatable withthe fibrinolytic is pulmonary embolus

Non-limiting examples of radioactive elements suitable for imaginginclude: ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁶⁴Cu, ⁹⁰Y, ¹⁶¹Tb, ¹⁷⁷Lu, and ¹¹¹In. Suchagents may be compelxed directly into the adrenomedullin molecule orrelated derivative or chelated to the adrenomedullin related peptidethrough a chelator selected from: diethylenetriaminepentaacetic acid(DTPA), 1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid(DOTA), 1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid(TETA), and 6-hydrazinonicotinamide (HYNIC), among others.

Imaging allows, for example, detecting the presence or absence ofpulmonary embolus in a mammal, for example in a human. To that effect alabelled adrenomedullin derivative is administered to the mammal in anamount and for a duration effective to achieve binding between thelabelled adrenomedullin derivative and pulmonaryadrenomedullin-receptor-bearing cells. Then, an image of thedistribution of the labelled adrenomedullin derivative in the lungs ofthe mammal is generated. Subsequently, the presence or absence ofpulmonary embolus is detected.

In a non-limiting example of implementation, the radiolabelledadrenomedullin derivative is administered to the mammal throughinjection of from about 0.1 nmol to about 100 nmol of 99mTc-AM-DTPA. Inanother non-limiting example of implementation, the radiolabelledadrenomedullin derivative is administered to the mammal throughinjection of from about 0.5 mCi to about 500 mCi of 99mTc-AM-DTPA.However, it is within the scope of the invention to inject any othersuitable amount of the labelled adrenomedullin derivative and toadminister the labelled adrenomedullin derivative using any othersuitable method.

In some embodiments of the invention, the labelled adrenomedullinderivative is detected to produce a model of the distribution oflabelled adrenomedullin in the lungs. Then, the model of the lungsindicates the presence of a likely pulmonary embolus through thepresence of a reduced activity region within the model. The reducedactivity region is a region of the model of the lungs wherein aconcentration of labelled adrenomedullin is substantially reduced withrespect to adjacent regions of the model of the lungs.

In other examples, the adrenomedullin derivative comprises an elementselected from: Fe, Ca, Mn, Mg, Cu, and Zn. These elements haveapplications, among other examples of application, to ion depletiontherapy for cancer and other pathologies. In these cases, non-limitativeexamples of suitable chelating agents used for binding the element toadrenomedullin include desferoxamine andN,N′,N″-tris(2-pyridylmethyl)-cis-1,3,5-triaminocyclohexane (tachpyr).

In other embodiments of the invention, a method of detecting thepresence and density of adrenomedullin receptor-bearing cells in amammal is provided. This method includes:

-   -   administering to the mammal a labelled adrenomedullin derivative        for a time and for a duration effective to achieve binding        between the labelled adrenomedullin derivative and        adrenomedullin-receptor-bearing cells, and    -   determining the distribution of the labelled adrenomedullin        derivative for a time and for a duration effective to obtain an        image of the mammal.

In other methods, at least one active agent is delivered to pulmonaryadrenomedullin-receptor-bearing cells in a mammal.

EXAMPLE 1 A Process to Produce ^(99m)Tc-Labelled AM

In this example, the adrenomedullin produced is a human adrenomedullinhaving the sequence:

(SEQ ID 1) H-Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser- Pro-Gln-Gly-Tyr-CONH₂

However, it is within the scope of the invention to use any othersuitable adrenomedullin, such as rat adrenomedullin or derivatives ofthe CT/CGRP/AMY peptide family, as well as their modified products suchas those obtained after N- and/or C-terminal substitution.

A method for synthesizing an a CT/CGRP/AMY peptide suitable for use withthe present invention, such as for example adrenomedullin, was performedas follows. The following commercial N-α-fluorenylmethyloxycarbonyl[Fmoc]-L-amino acids were used: Alanine [Fmoc-Ala],Arginine-N^(ω)-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)[Fmoc-Arg(Pbf)], Asparagine-NY-trityl [Fmoc-Asn(Trt)], Asparticacid-α-t-butyl ester [Fmoc-Asp(OtBu)], Cysteine-S-trityl[Fmoc-Cys(Trt)], Glutamine-N^(δ)-trityl [Fmoc-Gln(Trt)], Glycine[Fmoc-Gly], Histidine-N^(im)-trityl [Fmoc-His(Trt)], Isoleucine[Fmoc-Ile], Leucine [Fmoc-Leu], Lysine-N^(ε)-t-butyloxycarbonyl[Fmoc-Lys(Boc)], Methionine [Fmoc-Met], Phenylalanine [Fmoc-Phe],Proline [Fmoc-Pro], Serine-O-t-butyl [Fmoc-Ser(tBu)],Threonine-O-t-butyl [Fmoc-Thr(tBu)], Tyrosine-O-t-butyl [Fmoc-Tyr(tBu)]and Valine [Fmoc-Val].

Adrenomedullin and its CT/CGRP/AMY analogues were synthesized, using asolid phase procedure based on the Fmoc-amino acid chemistry-BOP reagent(benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate) coupling strategy. This procedure is betterdescribed in reference 35, which is hereby incorporated by reference.

In summary, a Fmoc-Rink-amide-acetamidonorleucylaminomethyl resin(4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy-acetamidonorleucylaminomethylresin) was used as the solid support. After a treatment with a 20%piperidine (Pip)-dimethylformamide (DMF) mixture, in order to remove theprotecting Fmoc moiety and free the amine anchor on the solid support,the first amino acid of the synthesis, corresponding to the last residueof the peptide sequence (Tyrosine), was coupled to the resin with BOPreagent, in the presence of diisopropylethylamine (DIEA). In function ofthe resin substitution, a ratio of 3 equivalents (eq) of Fmoc-aminoacid, 3 eq of BOP and 5 eq of DIEA was used for each coupling step andeach step was monitored using a ninhydrin test.

After the complete synthesis of the peptide chain, a final Fmocdeprotection step was carried out with 20% Pip/DMF. For derivativescontaining a N-terminal chelating functional, the resin-bound peptidewas transferred into a round-bottom flask and the incorporation of aN-substituting moiety (examples of such moieties include, but are notlimited to, diethylenetriaminepentaacetic acid (DTPA) or1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA) or1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA or6-hydrazinonicotinamide (HYNIC), among others) was achieved by mixingthe resin-adrenomedullin overnight, at 50° C., with 3 eq of thesubstituting compound, 3 eq of BOP reagent and 3 eq of DIEA dissolved ina solvent mixture of DMF-dichloromethane-dimethylsulfoxide (49%:49%:2%).

The peptide was finally cleaved from the resin using a mixture oftrifluoroacetic acid (TFA)-ethanedithiol (EDT)-phenol-H₂O (9.5 mL-0.25mL-0.3 g-0.25 mL; 10 mL/g of resin) for 2 hours, at room temperature.After TFA evaporation, the crude material was precipitated and washedusing diethylether. The peptide was then dried and kept at −20° C. untilcyclization and purification.

AM cyclization, purification and characterization was performed, asfollows. Crude adrenomedullin (AM) (200 mg) was dissolved in a 2 mLsolution of 50% dimethylsulfoxide/H₂O in order to generate the disulfidebridge between the cysteine side-chains. After 30 min at roomtemperature, the peptide solution was diluted with 500 mL of 10%acetonitrile (ACN) in aqueous TFA (0.06%) before being injected onto aRP-HPLC C₁₈ (15 μm; 300 Å) column (250×21.20 mm) (Jupiter column fromPhenomenex, Torrance, Calif.). The purification step was carried outusing a Waters Prep 590 pump system connected to a Waters Model 441absorbance detector. The flow rate was fixed at 20 mL/min and thepeptide was eluted with a solvent gradient of 0% to 100% solvent B, in 2h, where solvent A is 10% ACN in aqueous TFA (0.06%) and solvent B is45% ACN in aqueous TFA (0.06%).

The homogeneity of the various fractions was evaluated using analyticalRP-HPLC with a C₁₈ (5 μm; 300 Å) column (250×4.60 mm) ((Jupiter columnfrom Phenomenex, Torrance, Calif.) connected to a Beckman 128 solventmodule coupled to a Beckman 168 PDA detector. The flow rate was 1.0mL/min and the elution of the peptide was carried out with a lineargradient of 20 to 60% B, where A is TFA 0.06% and B is ACN. Aliquots of20 μl were injected and analyzed. Homogeneous fractions were pooled,lyophilized and then analyzed again by analytical HPLC, and by MALDI-TOFmass spectrometry (Voyager DE spectrometer—Applied Biosystems, FosterCity, Calif.) using a-cyano-4-hydroxycinnamic acid as a matrix forpeptide inclusion and ionization.

Labeling of a chelating adrenomedullin derivative (exemplified withDTPA-AM and ^(99m)technetium) was as follows. The substituted peptideDTPA-AM (18.5 μg-2.89 nmol) was dissolved in 1 mM HCl (100 μl) and then,SnCl₂.2H₂O (14.8 μl of a 0.2 mg/mL aqueous solution: 3 μg-13 nmol) wasadded, followed immediately with Na^(99m)TcO₄ (15 mCi-28.9 pmol) insaline. After 1 h at room temperature, the solution was diluted with 1mL of phosphate buffer-saline (PBS) at pH 7.4.

In other examples, adrenomedullin (AM) or AM fragments or AM analoguesare modified with agents able to bind radioactive and/or paramagneticchemical elements such as those from the following non limiting list:^(99m)technetium (^(99m)Tc), ¹¹¹indium (¹¹¹In), ⁶⁷gallium (⁶⁷Ga),⁶⁴copper (⁶⁴Cu), among others. In the present case, the modified AM, AMfragment or AM analogue is particularly suitable, but non-limitatively,for imaging with for instance, a gamma camera, a positron emissiontomography camera, a magnetic resonance instrument, or any othersuitable imaging device.

In other examples, adrenomedullin (AM) or AM fragments or AM analoguesare modified with agents able to bind radioactive elements such as thosefrom the following non exclusive list: ⁹⁰yttrium (⁹⁰Y), ¹⁶¹terbium(¹⁶¹Tb), ¹⁷⁷lutetium (¹⁷⁷Lu), ¹¹¹indium (¹¹¹In), among others. In thiscase, the modified AM, AM fragment or AM analogue are particularlysuitable, but non-limitatively, for application in radiotherapy.

In other examples, adrenomedullin (AM) or AM fragments or AM analoguesare modified with agents able to bind ions such as those produced fromthe elements appearing in the following non limiting list: iron (Fe),calcium (Ca), manganese (Mn), magnesium (Mg), copper (Cu), and zinc(Zn), among others. In the present case, the modified AM, AM fragment orAM analogues are particularly suitable, but non-limitatively, forapplication in chemotherapy, using, for example, an intracellular iondepletion strategy. In this particular case, possible chelating agentsfor ion depletion are selected form the following non-limiting list:desferioxamine, tachpyr(N,N′,N″-tris(2-pyridylmethyl)-cis-1,3,5-triaminocyclohexane), amongothers.

While a specific process for producing a labeled adrenomedullinderivative according to the invention is described hereinabove, thereader skilled in the art will readily appreciate that it is within thescope of the invention to produce labeled adrenomedullin derivativesthat are within the scope of the claimed invention in any other suitablemanner.

EXAMPLE 2 Pharmacokinetics of ¹²⁵I Labelled AM in Rats

Introduction

This example was designed to evaluate the biodistribution,pharmacokinetics and multiorgan clearance of AM in rats in vivo.Quantification of single-pass pulmonary kinetics of AM and its mechanismwas characterized further in dogs using the single bolusindicator-dilution technique.

Methods

All experimental procedures were performed in accordance with theregulations and ethical guidelines from Canadian Council for the Care ofLaboratory Animals, and received approval by the Animal Ethics andResearch Committee of the Montreal Heart Institute. Male Sprague-Dawleyrats (Charles River), weighing between 400-450 g, were anaesthetized byan initial intramuscular dose of xylazine (10 mg/kg of body weight) andketamine (50 mg/kg of body weight), followed by an intra-peritonealinjection of heparin (2000 units; Sigma). Catheters were inserted intothe right carotid artery and jugular vein. Heart rate and systemic bloodpressure were monitored continuously. Additional doses ofxylazine/ketamine were used if noxious stimuli (pinching the hind feet)elicited nociceptive motor reflexes or changes of the systemic bloodpressure. Venous and arterial blood samples (3 ml) were collected andcentrifuged (1875 g, 15 min, 4° C.) and the plasma saved for subsequentmeasurement of irAM (immunoreactive AM). A similar amount of saline wasinfused into the animals to prevent hypovolaemia.

Radiolabelled ¹²⁵I-rAM(1-50) (Amersham Biosciences) (¹²⁵I labelled ratadrenomedullin) was injected in a volume of 200 μl (0.3 μmol, 0.5 μCi)either into the right heart chambers via the right jugular vein catheter(n=10), or in the systemic circulation via the carotid catheter (n=10).A series of 200 μl blood samples were collected 1 min after the initialAM injection, then repeated every 5 min for a 30-min period. After eachcollection, an equal volume of saline was injected into the animal tomaintain blood volume and pressure. The animals were then killed and thelungs, liver, kidneys (en bloc with the adrenal glands) and heart wereremoved and gravity drained. The blood samples and organs were thenplaced in a gamma-counter (model 1470 Wizard; Wallac) to determine ¹²⁵Iradioactivity. Results are expressed as a percentage of totalradioactivity injected. Results for these experiments are shown in FIGS.1 and 2.

Effects of Antagonists on AM Clearance

Three groups of rats (n=20 in each) were studied and received either 200μl of hAM(22-52) (5.6 nmol; Bachem), 100 μl of CGRP (1.75 nmol; PhoenixPharmaceuticals) or 100 μl of unlabelled rAM(1-50) (17.5 nmol; AmericanPeptide). The drugs were given by either intraarterial (n=10 in eachgroup) or intravenous (n=10 in each group) injection 5 min before the¹²⁵I-rAM(1-50) bolus. Plasma samples and tissues were treated asdescribed above. Results are shown in FIG. 3.

Measurement of Endogenous rAM(1-50) Levels in Plasma and Tissues

Plasma levels were measured in samples obtained at baseline as describedabove (n=40). In order to evaluate endogenous tissue levels, tenadditional rats were studied. After being anaesthetized, the animalswere killed by removal of the lungs, liver, kidneys and heart.Homogenization of organs was performed by adding 2 ml of buffer [4 mol/lguanidine thiocyanate (Fisher Scientific) and 1% trifluoroacetic acid(Sigma)] to 200 mg of tissue samples with the use of an automaticrevolving pestle (DynaMix; Fisher Scientific). Homogenates werevortex-mixed and samples (100 μl) kept at 4 C for subsequent proteindetermination by Bradford analysis. Remaining samples were centrifugedat 1300 g (4 C) and the supernatant saved for processing. Tissues andplasma samples were extracted using Sep-Pak C18 cartridges (Waters) andirAM(1-50) was measured using a competitive RIA (PhoenixPharmaceuticals) according to the manufacturer's instructions. Thedetection limit of this assay is approx. 4.7 pg/tube with a specificityfor rAM(1-50) of 100%, without any cross reactivity (0%) with hAM(1-52),pro-hAM, pro-rAM, amylin and ET (endothelin)-1.

In Vivo Single-Pass Measurement of AM Clearance in Dogs

Dogs were anaesthetized and prepared as described in detail in reference[13], which is hereby incorporated by reference. A catheter was insertedinto the carotid artery and positioned just above the aortic valve. Thiscatheter was connected to a peristaltic pump for automated bloodwithdrawal. Another catheter was placed into the jugular vein andpositioned in the right ventricular outflow tract to allow bolusinjection of the study tracers. A bolus was prepared by adding 3.3 μCiof 125I-rAM(1-50) (2.9 μmol) to 3 ml of Evans-Blue-dye labelled albuminand 0.9% saline to give a final volume of 6 ml. The mixture wasseparated into three equal parts for the two successive experiments ineach animal and to realize dilution-curve standards. A baselinesingle-bolus indicator-dilution experiment was performed. After 5 min,CGRP (n=7), hAM(22-52) (n=8) and unlabelled rAM(1-50) (n=9) wereadministered as an intravenous bolus of 100 nmol and 5 min later asecond indicator dilution experiment was performed.

The collected samples were processed and indicator-dilution curvesconstructed and analyzed as described in previously cited reference 13.Cardiac output and mean tracer AM extraction during the pulmonarytransit time were computed from the curves. Mean tracer extractioncorresponded to the difference between the areas of the outflow curve ofthe vascular reference (albumin) and that of the extracted tracer (AM).Recirculation of the tracers apparent in the terminal portion of thecurves was removed by linear extrapolation of the semi-logarithmic downslopes. Results are shown in FIG. 4.

Statistical Analysis

Multiple group comparisons were performed by factorial ANOVA, followed,when a significant interaction was found, by the Bonferroni/Dunn t test.Plasma kinetics of 125I-rAM(1-50) was fitted using a two-phaseexponential decay equation with GraphPad Prism (version 4.0) software.Pulmonary clearance of 125I-AM(1-50) in rats after intravenous andintra-arterial injection was compared by two-tailed unpaired Student's ttest. Comparison between venous and arterial rAM(1-50) levels in plasmawas performed with a two-tailed paired Student's t test. In the canineexperiments, the effect of drugs on AM extraction was analyzed bytwo-tailed paired Student's t tests. A P value of <0.05 was consideredsignificant. All results are reported as means ±S.E.M.

Results

Kinetics of ¹²⁵I-rAM(1-50) in Plasma

As shown in FIG. 1, intravenously administered ¹²⁵IrAM(1-50) rapidlydecreased following a two-compartment model with a relatively rapiddistribution half-life of 2.0 min [95% Cl (confidence interval),1.98-2.01] and an elimination half-life of 15.9 min (95% Cl, 15.0-16.9).Compartmental analysis revealed that the ratio of rate constants forexchange between the central and peripheral compartments (k₁₋₂/k₂₋₁) wasrelatively high at 7.97, demonstrating an important distribution of druginto the peripheral compartment. The volumes of distribution werecomputed, including the volume of the central compartment (Vc=3.84 ml),the volume at steady state (Vss=12.5 ml) and the apparent volume ofdistribution (Varea=35 ml). Administration of a human AM fragment(hAM(22-52)), CGRP or unlabelled rAM(1-50) prior to the injection ofradiolabelled rAM(1-50) did not modify plasma kinetics, resulting inalmost perfectly superimposable curves (results not shown).

Biodistribution of 125I-rAM(1-50) After Infection

As shown in FIG. 2, the lungs predominantly retained the peptide 30 minafter the injected dose (P<0.001). There was proportionately only minorretention by the liver, kidneys and heart. Administration of hAM(22-52)and CGRP did not significantly modify this distribution, except inkidneys, where only hAM(22-52) elevated the retained activity (P<0.005).Injection of unlabelled rAM(1-50) caused an important reduction in lungactivity (P<0.001) and significantly increased (P<0.001) the amountretained by the liver, kidneys and heart.

Lung Retention After Intravenous Compared with Intra-ArterialAdministration

In the control group (n=10 per injection site), there was evidence ofimportant first-pass pulmonary retention with a more than 50% decline inthe amount of the peptide retained after intra-arterial compared withintravenous injection (FIG. 3). This pulmonary first-pass retention wasnot affected by prior administration of hAM(22-52) or CGRP. However,pre-treatment with rAM(1-50) did not decrease further the alreadylowered pulmonary retention after intra-arterial compared withintravenous injections.

Endogenous rAM(1-50) Levels in Plasma and Organs

There was no difference in irAM(1-50) levels in venous (3.1+−0.2 pmol/l)and arterial (3.2+−0.2 pmol/l) plasma (n=40). Tissue levels (n=10) weremore than 20-fold higher in the lungs (249.0+−48.3 pg/mg protein;P<0.001) compared with liver (11.1+−1.3 pg/mg of protein), kidneys(11.7+−1.4 pg/mg of protein) and heart (7.2+−0.9 pg/mg of protein).

Single-Pass Pulmonary Kinetics of ¹²⁵I-rAM(1-50) in Dogs In Vivo

Analysis of the indicator-dilution curve outflow profiles demonstrated asignificant first-pass retention of ¹²⁵IrAM(1-50). A typical experimentis shown in the FIG. 4(A). The curve for ¹²⁵I-rAM(1-50) progressivelydeviated from its vascular reference (labelled albumin). Therecirculation of tracers was removed by extrapolation of thesemi-logarithmic down slopes. The difference between the areas of thetwo tracers' curves, which represent mean tracer ¹²⁵I-rAM(1-50)extraction during a single pulmonary transit time, was 30% in thatexperiment. Plotting the natural log ratio of the two tracerscharacterized further the extraction over time (FIG. 4). Therelationship was found to be linear, demonstrating that extractionincreased over time with no evidence of return of the extracted peptideinto circulation. In terms of ordinary capillary modeling, the slope ofthis relationship represents the sequestration rate constant for¹²⁵I-rAM(1-50) by the lungs. In the same animal, a second experiment wasperformed after injection of unlabelled rAM(1-50) (FIG. 4B). There wasan evident reduction in pulmonary clearance with a smaller differentialcurve area compared with albumin (mean extraction 12%) and progressivelyconverging curves on the down slope. The log ratio is completelymodified with an initial plateau followed by a decrease, demonstratingthe return of the tracer into the circulation.

Mean single-pass pulmonary extraction of ¹²⁵IrAM(1-50) was 36.4±2.1%.This was significantly decreased (P<0.01) to 21.9±2.4% after theadministration of unlabelled rAM(1-50). Extraction was not affected byCGRP with 44.6±2.9% occurring in the control compared with 40.6±2.9%after administration. There was a slight but significant (P<0.01)decrease in extraction with hAM(22-52) from 40.0±1.7% before to31.4±3.3% after administration.

Discussion

In this study, plasma kinetics and biodistribution of exogenouslyadministered AM in rats as well as plasma and tissue levels ofendogenous AM were evaluated. Single-pass pulmonary clearance of AM indogs using the indicator-dilution technique was further quantified andcharacterized in vivo.

Injected AM has a relatively short elimination half-life of 16 min withrapid and important distribution into a peripheral compartment. Thelungs retain most of the injected activity with evidence of single-passclearance, since retention is lower after intra-arterial compared withintravenous injection. There was no difference in total endogenous irAMlevels across the pulmonary circulation with very high endogenous tissuelevels also found in the lungs compared with other organs. These datademonstrate that the lungs are a major site for AM clearance, theabsence of a gradient suggesting that the lungs also have the ability toproduce and release AM into the circulation.

A relatively small volume of distribution for the central compartment(3.5 ml) was found, which is, in fact, less than the total blood volumeof the rat. This is consistent with the very rapid clearance of AM fromplasma with evidence of a first-pass effect into the pulmonarycirculation. Thus a substantial proportion of intravenously injected AMis relatively rapidly cleared as it passes through the pulmonarycirculation and does not distribute into the systemic circulation. Theimportance of a first-pass pulmonary clearance was confirmed andquantified by the use of the indicator-dilution experiments in dogswhere it was found that approximately 36% of the injected AM wasretained within the few seconds of a single pulmonary transit time. Theoutflow profile demonstrates that the retained AM is bound to itsclearance site and does not return into the circulation. This, combinedwith the data in rats, would suggest that AM binds with relatively highaffinity and relatively irreversibly to its receptor. This is consistentwith previous data demonstrating important specific AM binding sites inthe lungs of rats and humans [15, 16], with maximum binding in the lungsbeing higher than in any other organ studied [16]. This profile isreminiscent of the potent vasoconstrictor ET-1, which is alsopredominantly cleared by the pulmonary circulation by the endothelialETB receptor [13].

The effects of AM are mediated by at least two different receptors [17].One is the CGRP receptor to which AM binds with low affinity, whereasthe other is considered a specific AM receptor that can blocked by theC-terminal fragment of AM, hAM(22-52). In the present study, it wasfound that ¹²⁵I-rAM(1-50) clearance by the lungs can be competitivelyinhibited by the administration of unlabelled rAM(1-50). Interestingly,however, unlabelled rAM(1-50) did not modify the plasma kinetics of thepeptide, as we observed a compensatory increase in retention by theliver, kidney and heart. This supports further the important clearancerole of the lungs and suggests that most of the injected unlabelled AMwas also retained by the lungs, explaining the lack of inhibition inperipheral organs where levels of ¹²⁵I-rAM(1-50) must have been higherthan those of the unlabelled peptide. There was no effect of similardoses of CGRP, demonstrating that the CGRP receptor is not responsiblefor pulmonary clearance. Administration of the C-terminal fragmenthAM(22-52) also did not modify pulmonary retention in rats, although itdid cause a small significant increase in the kidneys. These resultswere confirmed by the in vivo indicator-dilution studies in dogs wherewe found important first-pass extraction of 125I-rAM(1-50) which wasimportantly reduced after injection of unlabelled rAM(1-50), slightlyreduced after hAM(22-52) and unaffected by CGRP. Previous investigatorshave evaluated pulmonary clearance of AM in isolated rat lungs andpulmonary endothelial cells and found that AM levels in effluents andculture media were unchanged after CGRP, but increased afteradministration of hAM(22-52) [18]. The structural components of the CGRPand AM receptors, CRLR, RAMP1, RAMP2 and RAMP3 are all expressed in ratlungs [19]. Northern-blot analysis has revealed previously that RAMP2,which confers AM selectivity to the receptor, is highly expressed in ratlung tissues compared with RAMP1 and RAMP3 [20]. Using selective CRLRantibodies and immunohistochemistry, Hagner et al. [21,22] demonstratedintense staining in the alveolar capillaries of both humans and rats.These previous findings, together with the present study, suggest thatlung AM clearance is mediated by specific AM receptors, possibly at thelevel of the pulmonary vascular endothelium.

Conclusions

The lung is a primary site for AM clearance. There is importantfirst-pass pulmonary clearance of AM through specific receptors. Thissuggests that the lungs not only modulate circulating levels of thispeptide, but also represent its primary target.

EXAMPLE 3 Pharmacokinetics of 99mTc Labelled AM and Imaging Using Same

Widely accessible in most nuclear medical centres via ⁹⁹Mo/^(99m)Tcgenerator, technetium-99m shows suitable nuclear properties for nuclearimaging with γ-emitting of 140.5 keV and a short half-life of 6.01 h(34). To avoid strong perturbation of hAM1-52 chemical structure and,consequently, the loss of its biological properties duringradiolabelling with ^(99m)Tc, a successful procedure, called‘bifunctional approach’, has been proposed. This strategy consists oftethering a strong chelating group for the radionuclide to a point ofthe peptide that is irrelevant for preserving its biological properties(23, 26, 31). Thus, we developed chelated radiolabelled adrenomedullinderivatives, preferably a chelated hAM1-52 derivative usingdiethylenetriaminepentaacetic acid (DTPA) radiolabelled with ^(99m)Tc.

The present example was designed to systematically evaluate thebiodistribution, pharmacokinetics and multi-organic clearance of^(99m)Tc-DTPA-hAM1-52 in dogs in vivo. Furthermore, the purpose of thisinvestigation was to assess the utility of the radiolabelled peptide asa pulmonary vascular imaging agent.

Anesthesia and Animal Preparation

All experimental procedures were performed in accordance withregulations and ethical guidelines from Canadian Council for the Care ofLaboratory Animals, and received approval by the animal ethics andresearch committee of the Montreal Heart Institute. Mongrel dogsweighing between 20-30 kg and presenting negative Dirofilaria imitisblood test results were anesthetized by an initial intravenous dose ofpentobarbital sodium (50 mg/kg). Animals were intubated and mechanicallyventilated using room air. Cutaneous electrocardiographic leads wereinstalled, and 18F cathlon with three-way was installed on bothsaphenous vein for 0.9% sodium chloride perfusion, radiolabelledinjection and blood collection. A right arterial femoral catheter wasalso inserted using the Seldinger technique for continuous bloodpressure monitoring. Additional doses of pentobarbital sodium were usedif noxious stimuli (pinching near the eye) could elicit nociceptivemotor reflexes or changes of the systemic blood pressure.

Dogs (n=10) undergoing surgical procedures were anesthetized andprepared as previously described, but maintained ventilated with 1-3%isoflurane. Pulmonary lobectomy was obtained by performing a surgicalligature of the right median lobe of the lungs.

Pharmacokinetics of 99mTc-DTPA-hAM1-52 in Plasma

Purified and buffered ^(99m)Tc-DTPA-hAM1-52 samples were injected inright saphenous vein (n=6). A series of 2 mL blood samples werecollected 1 min after the initial AM injection for a 10-min period, thenrepeated every 5 min for the following 50-min period. Blood samples weretaken via left saphenous vein. After each collection, an equal volume ofsaline was injected into the animal to maintain blood volume andpressure. The blood samples were then placed in an automatic gammacounter (model 1272 Clinigamma, LKB Wallac, Finland) to determine^(99m)Tc activity. Results were expressed as a percentage of totalradioactivity injected per mL and are shown in FIG. 5.

Biodistribution of 99mTc-DTPA-hAM1-52 and Multi-Organic Clearance InVivo

Multi-organic biodistribution of ^(99m)Tc-DTPA-hAM1-52 was evaluatedwith an Anger camera (420/550 Mobile Radioisotope Gamma Camera;Technicare, Solon, Ohio, USA) equipped with on board computer, and alow-energy parallel-hole collimator (model 14S22014). Followingintravenous injection of ^(99m)Tc-DTPA-hAM1-52, dynamic acquisition ofthe lungs, heart, liver and kidneys was recorded for a 30-min period(one frame/sec during the first minute, then one frame/min for theremaining time). Static acquisitions was also recorded for wholeindividual organs, including lungs, kidneys, liver, heart, bladder,gallbladder and muzzle, at 30, 60, 120, 240 minutes after initialinjection. These recordings were performed both in ventral and dorsalpositions. Results are shown in FIGS. 6 and 7.

Gamma Camera Results Analysis

Dynamic and static acquisitions were evaluated by using Matlab version7.01 image analysis tools software. The ^(99m)Tc total count, ^(99m)Tcmean count, and region of interest (ROI) size were calculated for eachorgan. Data correction was applied for 1) radioactive decay, 2) surgicaltable attenuation (dorsal images only), 3) geometric mean, and 4)organ's attenuation based on transmission factor. Results were expressedas a percentage of total radioactivity injected and examples of imagesobtained are shown in FIGS. 8 and 9.

Statistical Analysis

Plasma kinetics of ^(99m)Tc-DTPA-hAM1-52 was fitted using a two-phaseexponential decay equation with GraphPad Prism version 4.0 software.Time effects on each organ biodistribution were analyzed by two-wayrepeated measures ANOVA followed, when a significant interaction wasfound, by Bonferroni/Dunn t-test. Multiple organs biodistributioncomparison at 30 minutes was performed by one-way ANOVA followed, when asignificant interaction was found, by Bonferroni/Dunn t-test. A P valuesof <0.05 was considered significant. All results are reported as mean±S.D.

Results

Kinetics of ^(99m)Tc-DTPA-hAM1-52 in Plasma (FIG. 5)

Intravenously administered ^(99m)Tc-DTPA-hAM1-52 decreased relativelyrapidly following a two-compartment model with a relatively rapiddistribution half-life of 1.75 min (95% confidence interval, Cl:1.31-2.65) and an elimination half-life of 42.14 min (Cl: 30.41-68.63).Compartmental analysis reveals that the ratio of rate constant forexchange between the central and peripheral compartments (k₁₋₂/k₂₋₁) isrelatively high at 24.09, demonstrating an important distribution ofdrug into the peripheral compartment.

Biodistribution of ^(99m)Tc-DTPA-hAM1-52 After Injection (FIG. 6)

The lungs predominantly retained the peptide with 27.00±2.76% of theinjected dose after 30 minutes (P<0.001), as compared to kidneys(19.17±3.06%), liver (11.67±1.37%), heart (7.17±2.04%), bladder(5.67±1.75%), gallbladder (0.96±0.38%), and muzzle (1.17±0.41%). Lungretention was mildly reduced with time but sustained up to 4 hours afterthe injection (15.83±2.32%). Furthermore, uptake progressively increasedin the bladder (26.83±4.36%) and gallbladder (0.83±0.41%), consequentlyto the excretion of the radiolabelled peptide. The ^(99m)Tc-DTPA-hAM1-52biodistribution in the kidneys, liver, and muzzle remained unchangedwith time, with respectively 20.67±1.51%, 10.67±1.75%, and 0.83±0.41% at240 minutes after peptide injection.

Dynamic Biodistribution ^(99m)Tc-DTPA-hAM1-52 After Injection (FIG. 7)

Analysis of dynamic multi-organic biodistribution demonstratessignificant pulmonary first pass retention of ^(99m)Tc-DTPA-hAM1-52. Thecurve for lungs clearance also shows recirculation of the radiolabelledpeptide, followed by a slow decrease with time. Moreover, heart curveindicates similar first pass retention of ^(99m)Tc-DTPA-hAM1-52, withouthowever sustained clearance with time. On the opposite, liver andkidneys dynamic biodistribution demonstrate only slow but continuousretention with time.

Selective Pulmonary Lobectomy Effects on ^(99m)Tc-DTPA-hAM1-52 Perfusion(FIGS. 8 and 9)

Homogeneous distribution of the tracer is evident in the lungs of anormal animal (FIG. 8) with substantially no detectable activity overthe region of the heart and little activity in the abdomen. This allowsfor good lung imaging without significant contaminant activity fromsurrounding organs. After surgical lobectomy mimicking the pathologiccondition of a pulmonary embolus (FIG. 9), there is an evident perfusiondefect which allows the diagnosis by external imaging. FIG. 9 showsimages obtained through anterior (panel A) and oblique (panel B) views.The perfusion defect was substantially wedge-shaped. This defect isindicated by an arrow and substantially delimitated by dotted lines

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

All references cited and/or discussed in this specification areincorporated herein by reference in their entirety and to the sameextent as if each reference was individually incorporated by reference.

The in vivo experiments in rats and dogs, as described in thespecification, may be predictive of biological effects in humans orother mammals and/or may serve as animal models for use of the presentinvention in humans or other mammals.

REFERENCES

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1. An adrenomedullin derivative comprising: an adrenomedullin peptidechelated to at least one active agent.
 2. The adrenomedullin derivativeof claim 1, wherein said adrenomedullin peptide comprises adrenomedullinhaving the sequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:1) or a fragment thereof.
 3. (canceled)
 4. (canceled)
 5. Theadrenomedullin derivative of claim 4, wherein said active agentcomprises a radioactive element selected from: ^(99m)Tc, ¹¹¹In, ⁶⁷Ga,⁶⁴Cu, ⁹⁰Y, ¹⁶¹Tb ¹⁷⁷Lu and ¹¹¹In.
 6. The adrenomedullin derivative ofclaim 5, wherein said adrenomedullin peptide is chelated to theradioactive element through a chelator selected from:diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA),1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),and 6-hydrazinonicotinamide (HYNIC).
 7. The adrenomedullin derivative ofclaim 6, wherein said radioactive element is ^(99m)Tc and said chelatoris diethylenetriaminepentaacetic acid (DTPA).
 8. The adrenomedullinderivative of claim 2, wherein said active agent comprises an elementselected from: Fe, Ca, Mn, Mg, Cu, and Zn.
 9. The adrenomedullinderivative of claim 8, wherein said active agent is complexed to saidadrenomedullin peptide through a chelating agent selected from:desferioxamine andN,N′,N″-tris(2-pyridylmethyl)-cis-1,3,5-triaminocyclohexane (tachpyr).10. The adrenomedullin derivative of claim 1, wherein said at least oneactive agent is selected from: active agents comprising at least oneparamagnetic element, active agents comprising at least one radioactiveelement, and fibrinolytic agents.
 11. A method of detecting the presenceor absence of pulmonary embolus in a mammal comprising: administering tosaid mammal a labelled adrenomedullin derivative in an amount and for aduration effective to achieve binding between the labelledadrenomedullin derivative and pulmonary adrenomedullin-receptor-bearingcells, the labelled adrenomedullin derivative being chelated to anactive agent comprising a radioactive element; generating an image ofthe distribution of the labelled adrenomedullin derivative in the lungsof said mammal; and detecting the presence or absence of pulmonaryembolus.
 12. (canceled)
 13. The method of claim 11, wherein said mammalis human.
 14. The method of claim 11, wherein said labelledadrenomedullin derivative comprises adrenomedullin having the sequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gin-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:1) or a fragment thereof.
 15. The method of claim 14, whereinsaid radioactive element is selected from: ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁶⁴Cu,and ⁹⁰Y.
 16. The method of claim 15, wherein said radioactive element is^(99m)Tc.
 17. The method of claim claim 16, wherein said labelledadrenomedullin derivative is chelated to said active agent throughdiethylenetriaminepentaacetic acid (DTPA).
 18. The method of claim 17,wherein said labelled adrenomedullin derivative is administered to saidmammal at a substantially hemodynamically inactive dose.
 19. The methodof claim 18, wherein said labelled adrenomedullin derivative isadministered to said mammal through injection of from about 0.5 mCi toabout 500 mCi of labelled adrenomedullin derivative.
 20. The method ofclaim 11, wherein said labelled adrenomedullin derivative is dissolvedinto a buffer solution and then administered to said mammal by injectioninto the bloodstream of the mammal.
 21. A method of detecting thepresence and density of adrenomedullin receptor-bearing cells in amammal comprising: administering to said mammal a labelledadrenomedullin derivative for a time and under conditions effective toachieve binding between the labelled adrenomedullin derivative andadrenomedullin-receptor-bearing cells, and determining the distributionof the labelled adrenomedullin derivative for a time and underconditions effective to obtain an image of said mammal.
 22. A method ofdelivering at least one active agent to pulmonaryadrenomedullin-receptor-bearing cells in a mammal, comprisingadministering to said mammal an adrenomedullin derivative chelated tothe active agent in an amount and for a duration effective to achievebinding between the adrenomedullin derivative and the pulmonaryadrenomedullin-receptor-bearing cells.
 23. The method of claim 22,wherein said mammal is a human.
 24. The method of claim 22, wherein thelabelled adrenomedullin derivative comprises adrenomedullin having thesequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-GIn-Gly-Tyr(SEQ ID NO:1) or a fragment thereof.
 25. The method of claim 24, whereinsaid at least one active agent is selected from: agents comprising atleast one radioactive element, agents comprising at least oneparamagnetic element, and fibrinolytic agents.
 26. The method of claim26, wherein said radioactive element is selected from: ^(99m)Tc, ¹¹¹In,⁶⁷Ga, ⁶⁴Cu, and ⁹⁰Y.
 27. The method of claim 27, wherein saidradioactive element is ^(99m)Tc.
 28. The method of claim 25, whereinsaid adrenomedullin derivative is chelated to ^(99m)Tc throughdiethylenetriaminepentaacetic acid (DTPA).
 29. The method of claim 25,wherein said radioactive element is suitable for imaging the lungs ofthe mammal or for radiotherapy.
 30. (canceled)
 31. (canceled) 32.(canceled)
 33. (canceled)
 34. (canceled)
 35. (canceled)
 36. The methodof claim 11, wherein said labelled adrenomedullin derivative is detectedto produce a model of the distribution of labelled adrenomedullin in thelungs.
 37. The method of claim 36, wherein said model of the lungsindicates the likely presence of a pulmonary embolus through thepresence of a reduced activity region within the model, said reducedactivity region being a region of the model of the lungs wherein aconcentration of labelled adrenomedullin is substantially reduced withrespect to adjacent regions of the model of the lungs.